Ion exchange chromatography of proteins and peptides

ABSTRACT

The present invention relates to an ion exchange chromatography process for purifying a peptide from a mixture containing the peptide and related impurities, and to an industrial method including such ion exchange chromatography process.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of Ser. No. 09/522,694 filed Mar. 10,2000, now U.S. Pat. No. 6,451,987 and claims priority under 35 U.S.C.119 of U.S. provisional application Nos. 60/125,882 and 60/179,335 filedMar. 24, 1999 and Jan. 31, 2000, respectively, and of Danish applicationnos. 1999 00360 and 2000 00083 filed Mar. 15, 1999 and Jan. 19, 2000,respectively, the contents of which are fully incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an ion exchange chromatography processfor purifying a peptide from a mixture containing said peptide andrelated impurities and to an industrial including such ion exchangechromatography process.

2. Description of the Related Art

For the purification and analysis of proteins and peptides,chromatography is a well-known and widely used method. A number ofdifferent chromatographic principles are applied, among these ionexchange chromatography (IEC). The IEC principle includes two differentapproaches: anion exchange and cation exchange according to the chargeof the ligands on the ion exchange resin. A conventional IECpurification process usually consists of one or more: equilibrationsections, application or loading sections, wash sections, elutionsections, and regeneration sections (cf. Remington's PharmaceuticalSciences, Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, orRemington: The Science and Practice of Pharmacy, 19th Edition (1995)).

The main principle of elution in IEC in industrial purificationprocesses is salt component gradients in an aqueous buffer solution atconstant pH, either as step or linear gradients (cf. S. Bjørn and L.Thim, Activation of Coagulation Factor VII to VIIa, Res. Discl. No. 269,564–565, 1986). Isocratic elution is possible, but seldom used. Organicsolvents or modifiers have occasionally been added to the solutions tokeep the protein or peptide on the desired form or just in solution (cf.K. H. Jørgensen, Process for Purifying Insulin, U.S. Pat. No. 3,907,676,Sep. 23, 1975; and J. Brange, O. Hallund and E. Sørensen, ChemicalStability of Insulin 5. Isolation, Characterisation and Identificationof Insulin Transformation Products, Acta Pharm. Nord. 4(4), 223–232,1992). Also, the change in pH may occasionally be employed to elute thetarget protein (cf. J. Lamy, J. Lamy, J. Weill, Arch. Biochem. Biophys.193, 140–149, 1979).

BRIEF SUMMARY OF THE INVENTION

In contrast to the above described IEC techniques for purification ofany protein or peptide, consisting of one or more equilibration steps,application or loading steps, wash steps, elution steps, andregeneration steps, the present invention relates to a novel elutiontechnique which is the combination of elution in a solution comprisingan organic modifier with the subsequent elution in an aqueous solutionat the same or a different pH optionally followed by a regenerationstep. The equilibration solution and the sample for application may ormay not contain the organic modifier. The elution of the peptide occursat non-denaturing conditions (in a solution free of organic modifier).Moreover, the elution of the peptide is performed in a single peak.

Accordingly, in a broad aspect the present invention relates to a cationexchange chromatography process for purifying a peptide from a mixturecomprising said peptide and related impurities, comprising the steps of:

a) eluting said related impurities of said mixture in a solutioncomprising an organic modifier, water, optionally a salt component andoptionally a buffer, at a linear or step gradient or isocratically insalt component, and at pH-values optionally maintained with a buffer sothat said peptide has a positive local or overall net charge and saidrelated impurities have a local or overall positive net charge which islower than the positive net charge of said peptide so as to remove saidrelated impurities,b) subsequently, eluting said peptide by a step or linear change to anaqueous solvent optionally with a salt component, at the same or higherpH-values optionally maintained with a buffer.

In another broad aspect the present invention relates to a cationexchange chromatography process for purifying a peptide from a mixturecomprising said peptide and related impurities, comprising the steps of:

a) eluting said related impurities of said mixture in a solutionconsisting essentially of an organic modifier, water, optionally a saltcomponent and optionally a buffer, at a linear or step gradient orisocratically in salt component, and at pH-values optionally maintainedwith a buffer so that said peptide has a positive local or overall netcharge and said related impurities have a local or overall positive netcharge which is lower than the positive net charge of said peptide so asto remove said related impurities,b) subsequently, eluting said peptide by a step or linear change to anaqueous solvent optionally with a salt component, at the same or higherpH-values optionally maintained with a buffer.

In another broad aspect the present invention relates to an anionexchange chromatography process for purifying a peptide from a mixturecomprising said peptide and related impurities, comprising the steps of:

a) eluting said related impurities of said mixture in a solutioncomprising an organic modifier, water, optionally a salt component andoptionally a buffer, at a linear or step gradient or isocratically insalt component, and at pH-values optionally maintained with a buffer sothat said peptide has a negative local or overall net charge and saidrelated impurities have a local or overall negative net charge which islower than the negative net charge of said peptide so as to remove saidrelated impurities,b) subsequently, eluting said peptide by a step or linear change to anaqueous solvent optionally with a salt component, at the same or lowerpH-values optionally maintained with a buffer.

In another broad aspect the present invention relates to an anionexchange chromatography process for purifying a peptide from a mixturecomprising said peptide and related impurities, comprising the steps of:

a) eluting said related impurities of said mixture in a solutionconsisting essentially of an organic modifier, water, optionally a saltcomponent and optionally a buffer, at a linear or step gradient orisocratically in salt component, and at pH-values optionally maintainedwith a buffer so that said peptide has a negative local or overall netcharge and said related impurities have a local or overall negative netcharge which is lower than the negative net charge of said peptide so asto remove said related impurities,b) subsequently, eluting said peptide by a step or linear change to anaqueous solvent optionally with a salt component, at the same or lowerpH-values optionally maintained with a buffer.

In the above aspects of the present process the elution in step a) couldalso be considered a washing step of related impurities.

The elution of the peptide in step b) occurs at non-denaturingconditions (in a solution free of organic modifier). Thus, the peptideis eluted to an aqueous solution with a solution comprising water andoptionally a salt component, an acid or base, and/or a buffer, butwithout the presence of an organic modifier.

In one embodiment of the present invention the ratio of organic modifierto water, on a weight percent basis, is from 1:99 to 99:1, such as from1:99 to 80:20, 20:80 to 80:20, 30:70 to 70:30, 35:50 to 50:35, or 40:50to 50:40. Each of these ranges constitutes an alternative embodiment ofthe present invention.

In further embodiments of the present invention the organic modifier isselected from C₁₋₆-alkanol, C₁₋₆-alkenol or C₁₋₆-alkynol, urea,guanidine, or C₁₋₆-alkanoic acid, such as acetic acid, C₂₋₆-glycol,C₃₋₇-polyalcohol including sugars, preferably C₁₋₆-alkanol andC₂₋₆-glycol, more preferably methanol, ethanol, propanols and butanolsand hexyl glycols, most preferably ethanol and 2-propanol. Each of theseorganic modifiers constitutes an alternative embodiment of the presentinvention.

In a further embodiment of the present invention the salt component instep a) is selected from any organic or inorganic salt and mixturesthereof, preferably NaCl, KCl, NH₄Cl, CaCl₂, sodium acetate, potassiumacetate, ammonium acetate, sodium citrate, potassium citrate, ammoniumcitrate, sodium sulphate, potassium sulphate, ammonium sulphate, calciumacetate or mixtures thereof, most preferred sodium acetate, potassiumacetate, ammonium acetate, NaCl, NH₄Cl, KCl. Each of these saltcomponents constitutes an alternative embodiment of the presentinvention.

In a further embodiment of the present invention the gradient in saltcomponent in step a) is a step gradient in the salt component.

In a further embodiment of the present invention the salt component instep a) is present in a step concentration selected from the range of0.1 mmol/kg to 3000 mmol/kg, preferably 1 mmol/kg to 1000 mmol/kg, morepreferably 5 mmol/kg to 500, most preferably 20 mmol/kg to 300 mmol/kg.Each of these ranges constitutes an alternative embodiment of thepresent invention.

In a further embodiment of the present invention the salt componentgradient in step a) is a linear gradient in salt component.

In a further embodiment of the present invention the salt component instep a) is present in a linear concentration selected from 0.1 mmol/kgto 3000 mmol/kg, preferably 1 mmol/kg to 1000 mmol/kg, more preferably 5mmol/kg to 500, most preferably 20 mmol/kg to 300 mmol/kg. Each of theselinear concentrations constitutes an alternative embodiment of thepresent invention.

In a further embodiment of the present invention no salt component ispresent in step a.

In a further embodiment of the present invention the salt component instep b) is selected from any organic or inorganic salt, preferably NaCl,KCl, NH₄Cl, CaCl₂, sodium acetate, potassium acetate, ammonium acetate,sodium citrate, potassium citrate, ammonium citrate, sodium sulphate,potassium sulphate, ammonium sulphate, calcium acetate or mixturesthereof, most preferred sodium acetate, potassium acetate, ammoniumacetate, NaCl, NH₄Cl, KCl. Each of these salt components constitutes analternative embodiment of the present invention.

In a further embodiment of the present invention the salt component instep b) is present in a concentration selected from the range of 0.1mmol/kg to 3000 mmol/kg, preferably 1 mmol/kg to 1000 mmol/kg, morepreferably 5 mmol/kg to 500, most preferably 20 mmol/kg to 300 mmol/kg.Each of these ranges constitutes an alternative embodiment of thepresent invention.

In a further embodiment of the present invention no salt component ispresent in step b).

In a further embodiment of the present invention the buffer in step a)or b) is independently selected from citrate buffers, phosphate buffers,tris buffers, borate buffers, lactate buffers, glycyl glycin buffers,arginine buffers, carbonate buffers, acetate buffers, glutamate buffers,ammonium buffers, glycin buffers, alkylamine buffers, aminoethyl alcoholbuffers, ethylenediamine buffers, tri-ethanol amine, imidazole buffers,pyridine buffers and barbiturate buffers and mixtures thereof,preferably citric acid, sodium citrate, sodium phosphate, phosphoricacid, glutamic acid, sodium glutamate, glycin, sodium carbonate,potassium citrate, potassium phosphate, potassium glutamate, potassiumcarbonate, tris-hydroxymethyl amino methane and boric acid and mixturesthereof. Each of these buffers constitutes an alternative embodiment ofthe present invention.

In a further embodiment of the present invention the buffer in step a)is present in a concentration selected from the range of 0.1 mmol/kg to500 mmol/kg, preferably 1 mmol/kg to 200 mmol/kg, more preferably 5mmol/kg to 100 mmol/kg, most preferably 10 mmol/kg to 50 mmol/kg. Eachof these ranges constitutes an alternative embodiment of the presentinvention.

In a further embodiment of the present invention the buffer in step b)is present in a concentration selected from the range of 0.1 mmol/kg to1000 mmol/kg, preferably 1 mmol/kg to 400 mmol/kg, most preferably 50mmol/kg to 200 mmol/kg. Each of these ranges constitutes an alternativeembodiment of the present invention.

In a further embodiment of the present invention no buffer is present instep a).

In a further embodiment of the present invention no buffer is present instep b).

In a further embodiment of the present invention the peptide to bepurified is selected from polypeptides, oligopeptides; proteins,receptors, vira, as well as homologues, analogues and derivativesthereof, preferably glucagon, hGH, insulin, aprotinin, FactorVII, TPA,FactorVIIa, FFR-FactorVIIa, heparinase, ACTH, Heparin Binding Protein,corticotropin-releasing factor, angiotensin, calcitonin, insulin,glucagon-like peptide-1, glucagon-like peptide-2, insulin-like growthfactor-1, insulin-like growth factor-2, fibroblast growth factors,gastric inhibitory peptide, growth hormone-releasing factor, pituitaryadenylate cyclase activating peptide, secretin, enterogastrin,somatostatin, somatotropin, somatomedin, parathyroid hormone,thrombopoietin, erythropoietin, hypothalamic releasing factors,prolactin, thyroid stimulating hormones, endorphins, enkephalins,vasopressin, oxytocin, opiods, DPP IV, interleukins, immunoglobulins,complement inhibitors, serpin protease inhibitors, cytokines, cytokinereceptors, PDGF, tumor necrosis factors, tumor necrosis factorsreceptors, growth factors and analogues as well as derivatives thereof,more preferably glucagon, hGH, insulin, aprotinin, FactorVII,FactorVIIa, FFR-FactorVIIa, heparinase, glucagon-like peptide-1,glucagon-like peptide-2 and analogues as well as derivatives thereof,such as Val⁸GLP-1 (7-37), Thr⁸GLP-1 (7-37), Met⁸GLP-1(7-37),Gly⁸GLP-1(7-37), Val⁸GLP-1(7-36) amide, Thr⁸GLP-1(7-36) amide,Met⁸GLP-1(7-36) amide, Gly⁸GLP-1(7-36) amide, Arg³⁴GLP-1(7-37), humaninsulin, and B28IsoAsp insulin. Each of these peptides constitutes analternative embodiment of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chromatograph obtained as described in Example 1.

FIGS. 2–3 are chromatographs of the sample for application and theeluate obtained as described in Example 1.

FIG. 4 is a chromatograph obtained as described in Example 3.

FIG. 5 is a chromatograph obtained as described in Example 5.

FIG. 6 is a chromatograph obtained as described in Example 9.

FIG. 7 is a chromatograph obtained as described in Example 10.

FIG. 8 is a chromatograph obtained as described in Example 11.

FIG. 9 is a chromatograph obtained as described in Example 12.

FIG. 10 is a chromatograph obtained as described in Example 13.

FIG. 11 is a chromatograph obtained as described in Example 14.

FIG. 12 is a chromatograph obtained as described in Example 16.

As an example illustrating the present invention, histidine has apredominant positive net charge below pH˜6.5, thus for cation exchangethe wash or elution section with organic solvent or modifier could beperformed below pH 6.5 to remove a truncated form missing histidine, andsubsequent elution of the target protein or peptide in the aqueoussolvent could be performed above pH 6.5. As a second example, thecarboxyl group of the C-terminal amino acid has a predominant negativenet charge above pH˜3.1, thus for anion exchange the wash or elutionsection with organic solvent could be performed above pH 3.1 to remove aform extended to an amide, and subsequent elution of the target proteinor peptide in the aqueous solvent could be performed both above or belowpH 3.1. As a third example, aspartic acid has a predominant negative netcharge above pH˜4.4, thus for anion exchange the wash or elution sectionwith organic solvent could be performed above pH 4.4 to remove atruncated form missing aspartic acid, and subsequent elution of thetarget protein or peptide in the aqueous solvent could be performedbelow pH 4.4. As a fourth example, glutamic acid has a predominantnegative net charge above pH˜4.4, thus for anion exchange the wash orelution section with organic solvent could be performed above pH 4.4 toremove a truncated form missing glutamic acid, and subsequent elution ofthe target protein or peptide in the aqueous solvent could be performedbelow pH 4.4. As a fifth example, γ-carboxy glutamic acid has apredominant negative net charge above pH˜4.4, thus for anion exchangethe wash or elution section with organic solvent could be performedabove pH 4.4 to remove a form missing one or more γ-carboxy groups ofspecific glutamic acid residues, and subsequent elution of the targetprotein or peptide in the aqueous solvent could be performed below pH4.4. As a sixth example, the amino group of the N-terminal amino acidhas a predominant positive net charge below pH˜8.0, thus for cationexchange the wash or elution section with organic solvent could beperformed below pH 8.0 to remove a form extended with an acyl group, andsubsequent elution of the target protein or peptide in the aqueoussolvent could be performed both above or below pH 8.0. As a seventhexample, cysteine has a predominant negative net charge above pH˜8.5,thus for anion exchange the wash or elution section with organic solventcould be performed above pH 8.5 to remove a poorly folded form resultingin free cysteine residues, and subsequent elution of the target proteinor peptide in the aqueous solvent could be performed below pH 8.5. As aneighth example, tyrosine has a predominant negative net charge abovepH˜10.0, thus for anion exchange the wash or elution section withorganic solvent could be performed above pH 10.0 to remove a truncatedform missing a tyrosine residue, and subsequent elution of the targetprotein or peptide in the aqueous solvent could be performed below pH10.0. As a ninth example, lysine has a predominant positive net chargebelow pH˜10.0, thus for cation exchange the wash or elution section withorganic solvent could be performed below pH 10.0 to remove a formacylated in the side chain of the lysine residue, and subsequent elutionof the target protein or peptide in the aqueous solvent could beperformed above or below pH 10.0. As a tenth example, arginine has apredominant positive net charge below pH˜12.0, thus for anion exchangethe wash or elution section with organic solvent could be performedbelow pH 12.0 to remove a truncated form missing an arginine residue,and subsequent elution of the target protein or peptide in the aqueoussolvent could be performed above pH 12.0. (pK_(A)-values used in theseexamples are from: L. Stryer. Biochemistry, 3^(rd) edition, W. H.Freeman and Company, New York, Table 2-1 page 21).

Specific peptide examples of the above-mentioned method are separationof Arg³⁴GLP-1(7-37) and Arg³⁴GLP-1(9-37) by cation exchangechromatography, human insulin and B30 human insulin ethyl ester by anionexchange chromatography, B28IsoAsp insulin and DesB23-30 insulin byanion exchange chromatography, prothrombin and des-γ-carboxy-Gluprothrombin by anion exchange chromatography, Arg³⁴GLP-1 (7-37) andArg³⁴GLP-1(10-37) by anion exchange chromatography,Lys^(B29)-(N-ε(α-tetradecanoyl))-desB30 insulin and DesB30 insulin bycation exchange chromatography, Lys^(B29)-(N-ε(α-tetradecanoyl))-desB30insulin andLys^(B29)-(N-ε(α-tetradecanoyl))-A1-(N-ε(α-tetradecanoyl))-desB30insulin by cation exchange chromatography, aprotinin andDes-Arg-Pro-aprotinin by cation exchange chromatography, andGlucagon₍₁₋₂₉₎ and Glucagon₍₆₋₂₉₎ by anion exchange chromatography.

The peptides can be produced by a method which comprises culturing orfermenting a host cell containing a DNA sequence encoding thepolypeptide and capable of expressing the polypeptide in a suitablenutrient medium under conditions permitting the expression of thepeptide, after which the resulting peptide is recovered from the cultureor fermentation broth. Hereinafter, culturing will be used to cover bothculturing and fermenting and the like.

The medium used to culture the cells may be any conventional mediumsuitable for growing the host cells, such as minimal or complex mediacontaining appropriate supplements. Suitable media are available fromcommercial suppliers or may be prepared according to published recipes(e.g. in catalogues of the American Type Culture Collection). Thepeptide produced by the cells may then be recovered from the culturemedium by conventional procedures including, optionally lysis of cells,separating the host cells from the medium by centrifugation orfiltration, precipitating the proteinaceous components of thesupernatant or filtrate by means of a salt, e.g. ammonium sulphate,purification by conventional purification techniques, such aschromatographic techniques, if necessary, purification by ion exchangechromatography according to the present invention, and subsequently,subjecting to analytical tests, e.g. PAGE, IEF, if necessary, subjectingto further purification, if necessary, and isolation of the purepeptide.

During the recovery of the resulting peptide from the culture medium,but before purification by ion exchange chromatography according to thepresent invention, the mixture comprising the peptide and relatedimpurities may optionally be chemically modified by conventionaltechniques, e.g. by alkylation, acylation, ester formation or amideformation or the like.

The DNA sequence encoding the parent peptide may suitably be of genomicor cDNA origin, for instance obtained by preparing a genomic or cDNAlibrary and screening for DNA sequences coding for all or part of thepeptide by hybridisation using synthetic oligonucleotide probes inaccordance with standard techniques (see, for example, Sambrook, J,Fritsch, E F and Maniatis, T, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York, 1989). The DNA sequenceencoding the peptide may also be prepared synthetically by establishedstandard methods, e.g. the phosphoamidite method described by Beaucageand Caruthers, Tetrahedron Letters 22 (1981), 1859–1869, or the methoddescribed by Matthes et al., EMBO Journal 3 (1984), 801–805. The DNAsequence may also be prepared by polymerase chain reaction usingspecific primers, for instance as described in U.S. Pat. No. 4,683,202or Saiki et al., Science 239 (1988), 487–491.

The DNA sequence may be inserted into any vector which may convenientlybe subjected to recombinant DNA procedures, and the choice of vectorwill often depend on the host cell into which it is to be introduced.Thus, the vector may be an autonomously replicating vector, i.e. avector which exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g. a plasmid.Alternatively, the vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which the DNA sequenceencoding the peptide is operably linked to additional segments requiredfor transcription of the DNA, such as a promoter. The promoter may beany DNA sequence which shows transcriptional activity in the host cellof choice and may be derived from genes encoding proteins eitherhomologous or heterologous to the host cell. Examples of suitablepromoters for directing the transcription of the DNA encoding thepeptide of the invention in a variety of host cells are well known inthe art, cf. for instance Sambrook et al., supra.

The DNA sequence encoding the peptide may also, if necessary, beoperably connected to a suitable terminator, polyadenylation signals,transcriptional enhancer sequences, and translational enhancersequences. The recombinant vector of the invention may further comprisea DNA sequence enabling the vector to replicate in the host cell inquestion.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell or one whichconfers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin,chloramphenicol, neomycin, hygromycin or methotrexate.

To direct a peptide into the secretory pathway of the host cells, asecretory signal sequence (also known as a leader sequence, preprosequence or pre sequence) may be provided in the recombinant vector. Thesecretory signal sequence is joined to the DNA sequence encoding thepeptide in the correct reading frame. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the peptide. Thesecretory signal sequence may be that normally associated with thepeptide or may be from a gene encoding another secreted protein.

The procedures used to ligate the DNA sequences coding for the peptide,the promoter and optionally the terminator and/or secretory signalsequence, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al., supra).

The host cell into which the DNA sequence or the recombinant vector isintroduced may be any cell which is capable of producing the presentpeptide and includes bacteria, vira, e.g. baculo virus; yeast, fungi,insect cells and higher eukaryotic cells. Examples of suitable hostcells well known and used in the art are, without limitation, E. coli,Saccharomyces cerevisiae, or mammalian BHK or CHO cell lines.

Some of the peptides, in particular the oligopeptides, can be producedaccording to conventional organic peptide synthetic chemistry. Theresulting synthetic mixture may then be chemically modified, e.g. byalkylation, acylation, ester formation or amide formation or the like,and purified, or purified as it is and then modified chemically asmentioned above.

Preparation of Factor VIIa

Human purified factor VIIa suitable for use in the present invention ispreferably made by DNA recombinant technology, e.g. as described byHagen et al., Proc. Natl. Acad. Sci. USA 83: 2412–2416, 1986 or asdescribed in European Patent No. 200.421 (ZymoGenetics). Factor VIIaproduced by recombinant technology may be authentic factor VIIa or amore or less modified factor VIIa provided that such factor VIIa hassubstantially the same biological activity for blood coagulation asauthentic factor VIIa. Such modified factor VIIa may be produced bymodifying the nucleic acid sequence encoding factor VII either byaltering the amino acid codons or by removal of some of the amino acidcodons in the nucleic acid encoding the natural FVII by known means,e.g. by site-specific mutagenesis.

Factor VII may also be produced by the methods described by Broze andMajerus, J. Biol. Chem. 255 (4): 1242–1247, 1980 and Hedner and Kisiel,J. Clin. Invest. 71: 1836–1841, 1983. These methods yield factor VIIwithout detectable amounts of other blood coagulation factors. An evenfurther purified factor VII preparation may be obtained by including anadditional gel filtration as the final purification step. Factor VII isthen converted into activated FVIIa by known means, e.g. by severaldifferent plasma proteins, such as factor XIIa, IX a or Xa.Alternatively, as described by Bjoern et al. (Research Disclosure, 269September 1986, pp. 564–565), factor VII may be activated by passing itthrough an ion-exchange chromatography column, such as Mono Q®(Pharmacia fine Chemicals) or the like.

Modified or Inactivated FVIIa (FVIIai) May be Produced by the FollowingMethods:

International Application No. WO 92/15686 relates to modified FactorVIIa, polynucleic acid and mammalian cell lines for the production ofmodified Factor VIIa, and compositions comprising modified Factor VIIafor inhibiting blood coagulation.

Modified Factor VII may be encoded by a polynucleotide moleculecomprising two operatively linked sequence coding regions encoding,respectively, a pre-pro peptide and a gla domain of a vitaminK-dependent plasma protein, and a gla domain-less Factor VII protein,wherein upon expression said polynucleotide encodes a modified FactorVII molecule which does not significantly activate plasma Factors X orIX, and is capable of binding tissue factor.

The catalytic activity of Factor VIIa can be inhibited by chemicalderivatization of the catalytic centre, or triad. Derivatization may beaccomplished by reacting Factor VII with an irreversible inhibitor suchas an organophosphor compound, a sulfonyl fluoride, a peptide halomethylketone or an azapeptide, or by acylation, for example. Preferred peptidehalomethyl ketones include PPACK (D-Phe-Pro-Arg chloromethyl-ketone;(see U.S. Pat. No. 4,318,904, incorporated herein by reference),D-Phe-Phe-Arg and Phe-Phe-Arg chloromethylketone (FFR-cmk); and DEGRck(dansyl-Glu-Gly-Arg chloromethylketone).

The catalytic activity of Factor VIIa can also be inhibited bysubstituting, inserting or deleting amino acids. In preferredembodiments amino acid substitutions are made in the amino acid sequenceof the Factor VII catalytic triad, defined herein as the regions, whichcontain the amino acids, which contribute to the Factor VIIa catalyticsite. The substitutions, insertions or deletions in the catalytic triadare generally at or adjacent to the amino acids which form the catalyticsite. In the human and bovine Factor VII proteins, the amino acids,which form a catalytic “triad”, are Ser₃₄₄, AsP₂₄₂, and His₁₉₃(subscript numbering indicating position in the sequence). The catalyticsites in Factor VII from other mammalian species may be determined usingpresently available techniques including, among others, proteinisolation and amino acid sequence analysis. Catalytic sites may also bedetermined by aligning a sequence with the sequence of other serineproteases, particularly chymotrypsin, whose active site has beenpreviously determined (Sigler et al., J. Mol. Biol., 35:143–164 (1968),incorporated herein by reference), and there from determining from saidalignment the analogous active site residues.

In preferred embodiments of human and bovine Factor VII, the active siteresidue Ser₃₄₄ is modified, replaced with Gly, Met, Thr, or morepreferably, Ala. Such substitution could be made separately or incombination with substitution(s) at other sites in the catalytic triad,which includes His₁₉₃ and Asp₂₄₂.

The amino acids, which form the catalytic site in Factor VII, such asSer₃₄₄, Asp₂₄₂, and His₁₉₃ in human and bovine Factor VII, may either besubstituted or deleted. Within the present invention, it is preferred tochange only a single amino acid, thus minimizing the likelihood ofincreasing the antigenicity of the molecule or inhibiting its ability tobind tissue factor, however two or more amino acid changes(substitutions, additions or deletions) may be made and combinations ofsubstitution(s), addition(s) and deletion(s) may also be made. In apreferred embodiment for human and bovine Factor VII, Ser₃₄₄ ispreferably substituted with Ala, but Gly, Met, Thr or other amino acidscan be substituted. It is preferred to replace Asp with Glu and toreplace His with Lys or Arg. In general, substitutions are chosen todisrupt the tertiary protein structure as little as possible. The modelof Dayhoff et al. (in Atlas of Protein Structure 1978, Nat'l Biomed.Res. Found., Washington, D.C.), incorporated herein by reference, may beused as a guide in selecting other amino acid substitutions. One mayintroduce residue alterations as described above in the catalytic siteof appropriate Factor VII sequence of human, bovine or other species andtest the resulting protein for a desired level of inhibition ofcatalytic activity and resulting anticoagulant activity as describedherein. For the modified Factor VII the catalytic activity will besubstantially inhibited, generally less than about 5% of the catalyticactivity of wild-type Factor VII of the corresponding species, morepreferably less than about 1%.

The modified Factor VII may be produced through the use of recombinantDNA techniques.

The amino acid sequence alterations may be accomplished by a variety oftechniques. Modification of the DNA sequence may be by site-specificmutagenesis. Techniques for site-specific mutagenesis are well known inthe art and are described by, for example, Zoller and Smith (DNA3:479–488, 1984). Thus, using the nucleotide and amino acid sequences ofFactor VII, one may introduce the alteration(s) of choice. The modifiedFVIIa may also be produced by chemical methods.

FFR-FVIIa (that is, D-Phe-Phe-Arg-FVIIa)

EXAMPLE FFR Chloromethyl Ketone

Blockage of the Active Site of FVIIa with FFR Chloromethyl Ketone.

Blockage of the active site serine and histidine with chloromethylketone is a well-known method for irreversible inactivation of serineproteases. In order to optimise the blockage of a given protease it isimportant to choose a chloromethyl ketone derivative, which reactsspecifically with the active site and with a fast on-rate. Suchderivatives can be developed by attachment to the chloromethyl ketonegroup of an oligopeptide, which interacts, with the substrate-bindingpocket of the particular serine protease of interest.

Glutamyl-Glycyl-Arginine chloromethyl ketone (EGR-ck or its Dansylderivative, DEGR-ck) (S. Higashi, H. Nishimura, S. Fujii, K. Takada, S.Iwanaga, (1992) J. Biol. Chem. 267, 17990) or Prolyl-Phenyl-Argininechloromethyl ketone (PFR-ck) (J. H. Lawson, S. Butenas, K. Mann, (1992)J. Biol. Chem. 267, 4834; J. Contrino, G. A. Hair, M. A. Schmeizl, F. R.Rickles, D. L. Kreutzer (1994) Am. J. Pathol. 145, 1315) have beenapplied as active site inhibitors of FVIIa. Compared with thesechloromethyl ketones application of FFRck represents a rate increase of10–70 fold.

The specificity of the reaction with FFR-chloromethyl ketone derivativeof FVIIa was checked by HPLC and peptide mapping which showed that FVIIahad reacted with FFR-chloromethyl ketone in a 1:1 ratio such that >98%could be recovered as the expected product derivatized at histidine 193.

Inactivation of FVIIa by Various Chloromethyl Ketones:

3 μM FVIIa was incubated with 12 μM of chloromethyl ketone derivative in50 mM Tris HCl, 100 mM NaCl, 5 mM CaCl₂, 0.01% Tween-80, pH 7.4. Sampleswere withdrawn at various time intervals as indicated and and diluted 20times for activity measurements in 50 mM Tris HCl, 100 mM NaCl, 5 mMCaCl₂, 0.01% Tween-80, pH 7.4 containing 1 mM IIe-Pro-Arg-pNA. Theresidual FVIIa activity was measured by the increase in absorbance at405 nm.

Usually, the mixture comprising the peptide and related impurities to bepurified by ion exchange chromatography according to the presentinvention, will also contain amino acids, small peptides, largepeptides, unrelated proteins, reactants, cell debris, HCP, endotoxins,and/or vira depending on whether recombinant DNA techniques and/orchemical modification techniques have been used or whether organicpeptide synthetic chemistry techniques have been used.

Thus, any method, such as an industrial method, for producing a purepeptide, which includes an IEC process according to the presentinvention is also an aspect of the present application.

Accordingly, the present invention relates in a further aspect to anindustrial method for producing a pure peptide, the method including acation exchange chromatography process for purifying a peptide from amixture comprising said peptide and related impurities, comprising thesteps of:

a) eluting said related impurities of said mixture in a solutionconsisting essentially of an organic modifier, water, optionally a saltcomponent and optionally a buffer, at a linear or step gradient orisocratically in salt component, and at pH-values optionally maintainedwith a buffer so that said peptide has a positive local or overall netcharge and said related impurities have a local or overall positive netcharge which is lower than the positive net charge of said peptide so asto remove said related impurities,b) subsequently, eluting said peptide by a step or linear change to anaqueous solvent optionally with a salt component, at the same or higherpH-values optionally maintained with a buffer.

The present invention relates in a further aspect to a method forisolating a peptide, the method including purification of a peptide froma mixture comprising said peptide and related impurities via a cationexchange chromatography process, the cation exchange chromatographyprocess comprising the steps of:

-   -   a) eluting said related impurities of said mixture in a solution        comprising an organic modifier, water, optionally a salt        component and optionally a buffer, at a linear or step gradient        or isocratically in salt component, and at pH-values optionally        maintained with a buffer so that said peptide has a positive        local or overall net charge and said related impurities have a        local or overall positive net charge which is lower than the        positive net charge of said peptide so as to remove said related        impurities,    -   b) subsequently, eluting said peptide by a step or linear change        to an aqueous solvent optionally with a salt component, at the        same or higher pH-values optionally maintained with a buffer;        and subsequently, if necessary, subjecting to analytical tests        and/or further purification, and isolating said peptide in a        conventional manner.

The present invention relates in a further aspect to a method forisolating a peptide, the method including purification of a peptide froma mixture comprising said peptide and related impurities via a cationexchange chromatography process, the cation exchange chromatographyprocess comprising the steps of:

-   -   a) eluting said related impurities of said mixture in a solution        consisting essentially of an organic modifier, water, optionally        a salt component and optionally a buffer, at a linear or step        gradient or isocratically in salt component, and at pH-values        optionally maintained with a buffer so that said peptide has a        positive local or overall net charge and said related impurities        have a local or overall positive net charge which is lower than        the positive net charge of said peptide so as to remove said        related impurities,    -   b) subsequently, eluting said peptide by a step or linear change        to an aqueous solvent optionally with a salt component, at the        same or higher pH-values optionally maintained with a buffer;        and subsequently, if necessary, subjecting to analytical tests        and/or further purification, and isolating said peptide in a        conventional manner.

The present invention relates in a further aspect to an industrialmethod for producing a pure peptide, the method including an anionexchange chromatography process for purifying a peptide from a mixturecomprising said peptide and related impurities, comprising the steps of:

a) eluting said related impurities of said mixture in a solutionconsisting essentially of an organic modifier, water, optionally a saltcomponent and optionally a buffer, at a linear or step gradient orisocratically in salt component, and at pH-values optionally maintainedwith a buffer so that said peptide has a negative local or overall netcharge and said related impurities have a local or overall negative netcharge which is lower than the negative net charge of said peptide so asto remove said related impurities,

b) subsequently, eluting said peptide by a step or linear change to anaqueous solvent optionally with a salt component, at the same or lowerpH-values optionally maintained with a buffer.

The present invention relates in a still further aspect to a method forisolating a peptide, the method including purification of a peptide froma mixture comprising said peptide and related impurities via an anionexchange chromatography process, the anion exchange chromatographyprocess comprising the steps of:

-   -   a) eluting said related impurities of said mixture in a solution        comprising an organic modifier, water, optionally a salt        component and optionally a buffer, at a linear or step gradient        or isocratically in salt component, and at pH-values optionally        maintained with a buffer so that said peptide has a negative        local or overall net charge and said related impurities have a        local or overall negative net charge which is lower than the        negative net charge of said peptide so as to remove said related        impurities,    -   b) subsequently, eluting said peptide by a step or linear change        to an aqueous solvent optionally with a salt component, at the        same or lower pH-values optionally maintained with a buffer;        and subsequently, if necessary, subjecting to analytical tests        and/or further purification, and isolating said peptide in a        conventional manner.

The present invention relates in a still further aspect to a method forisolating a peptide, the method including purification of a peptide froma mixture comprising said peptide and related impurities via an anionexchange chromatography process, the anion exchange chromatographyprocess comprising the steps of:

-   -   a) eluting said related impurities of said mixture in a solution        consisting essentially of an organic modifier, water, optionally        a salt component and optionally a buffer, at a linear or step        gradient or isocratically in salt component, and at pH-values        optionally maintained with a buffer so that said peptide has a        negative local or overall net charge and said related impurities        have a local or overall negative net charge which is lower than        the negative net charge of said peptide so as to remove said        related impurities,    -   b) subsequently, eluting said peptide by a step or linear change        to an aqueous solvent optionally with a salt component, at the        same or lower pH-values optionally maintained with a buffer;        and subsequently, if necessary, subjecting to analytical tests        and/or further purification, and isolating said peptide in a        conventional manner.

Any possible combination of two or more of the embodiments describedherein, is comprised within the scope of the present invention.

The term “an organic modifier”, as used herein, is intended to includean organic solvent or organic compound soluble in water or mixturesthereof, which modifier induces a favorable and changed selectivitybetween the unwanted related impurity or impurities and the peptide andthe ion exchanger. Whether or not a selected modifier induces saidselectivity will usually depend on the related impurity or impurities,and may be tested by trial-and-error. The organic modifier includes butis not limited to C₁₋₆-alkanol, C₁₋₆-alkenol or C₁₋₆-alkynol, urea,guanidine•HCl, or C₁₋₆-alkanoic acid, such as acetic acid, C₂₋₆-glycol,C₃₋₇-polyalcohol including sugars or mixtures thereof.

The term “C₁₋₆-alkanol”, “C₁₋₆-alkenol” or “C₁₋₆-alkynol”, as usedherein, alone or in combination is intended to include those C₁₋₆alkane,C₁₋₆alkene or C₁₋₆alkyne groups of the designated length in either alinear or branched or cyclic configuration whereto is linked a hydroxyl(—OH) (cf. Morrison & Boyd, Organic Chemistry, 4^(th) ed). Examples oflinear alcohols are methanol, ethanol, n-propanol, allyl alcohol,n-butanol, n-pentanol and n-hexanol. Examples of branched alcohols are2-propanol and tert-butyl alcohol. Examples of cyclic alcohols are cyclopropyl alcohol and 2-cyclohexen-1-ol.

The term “C₁₋₆-alkanoic acid”, as used herein, is intended to include agroup of the formula R′COOH wherein R′ is H or C₁₋₅alkyl, such asacetic, propionic, butyric, ?-methylbutyric, or valeric acid (cf.Morrison & Boyd, Organic Chemistry, 4^(th) ed).

The term “C₁₋₅-alkyl”, as used herein, is intended to include a branchedor straight alkyl group having from one to five carbon atoms. TypicalC₁₋₅-alkyl groups include, but are not limited to, methyl, ethyl,n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl,isopentyl, and the like (cf. Morrison & Boyd, Organic Chemistry, 4^(th)ed).

The term “C₂₋₆-glycol”, as used herein, is intended to include aC₂₋₆-alkane containing two hydroxyl groups on different carbon atomswhich may be adjacent or not. A typical C₂₋₆-glycol include, but is notlimited to 1,2-ethanediol, 1,2-propanediol, or 2-methyl-2,4-pentanediol(cf. Morrison & Boyd, Organic Chemistry, 4^(th) ed).

The term “C₂₋₆-alkane”, as used herein, is intended to include abranched or straight alkane group having from two to six carbon atoms.Typical C₂₋₆-alkane groups include, but are not limited to ethane,propane, iso-propane, butane, iso-butane, pentane, hexane and the like(cf. Morrison & Boyd, Organic Chemistry, 4^(th) ed).

The term “C₃₋₇-polyalcohol including sugars”, as used herein, isintended to include a group of the formula HOCH₂(CHOH)_(n)CH₂OH whereinn is an integer from 1–5, and monosaccharides such as mannose, glucose(cf. Morrison & Boyd, Organic Chemistry, 4^(th) ed).

The term “peptide” or “peptides”, as used herein, is intended to includesuch polypeptides, oligopeptides, proteins, as well as homologues,analogues and derivatives thereof, which are capable of being producedby conventional recombinant DNA techniques as well as conventionalsynthetic methods. Such peptides include but are not limited toglucagon, hGH, insulin, aprotinin, FactorVII, TPA, FactorVIIa(NovoSeven®), available from Novo Nordisk A/S, Bagsvaerd, Denmark),FactorVIIai, FFR-FactorVIIa, heparinase, ACTH, corticotropin-releasingfactor, angiotensin, calcitonin, insulin, glucagon-like peptide-1,glucagon-like peptide-2, insulin-like growth factor-1, insulin-likegrowth factor-2, gastric inhibitory peptide, growth hormone-releasingfactor, pituitary adenylate cyclase activating peptides, secretin,enterogastrin, somatostatin, somatotropin, somatomedin, parathyroidhormone, thrombopoietin, erythropoietin, hypothalamic releasing factors,prolactin, thyroid stimulating hormones, endorphins, enkephalins,vasopressin, oxytocin, opiods, GIP, exendins, peptidehistidine-methionine amide, helospectins, helodermin, pituitaryadenylate cyclase activating peptide-related peptide, vasoactiveintestinal polypeptide and analogues thereof, wherein analogues is usedto designate a peptide wherein one or more amino acid residues of theparent peptide have been substituted by another amino acid residueand/or wherein one or more amino acid residues of the parent peptidehave been deleted and/or wherein one or more amino acid residues havebeen added to the parent peptide. Such addition can take place either atthe N-terminal end or at the C-terminal end of the parent peptide orboth. Derivatives are used in the present text to designate a peptide inwhich one or more of the amino acid residues of the parent peptide havebeen chemically modified, e.g. by alkylation, acylation, ester formationor amide formation or the like.

The term “salt component” as used herein, is intended to include anyorganic or inorganic salt, including but not limited to NaCl, KCl,NH₄Cl, CaCl₂, sodium acetate, potassium acetate, ammonium acetate,sodium citrate, potassium citrate, ammonium citrate, sodium sulphate,potassium sulphate, ammonium sulphate, calcium acetate or mixturesthereof (cf. Remington's Pharmaceutical Sciences, Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, or Remington: The Science andPractice of Pharmacy, 19th Edition (1995), or handbooks fromAmersham-Pharmacia Biotech).

The term “a buffer” as used herein, is intended to include any bufferincluding but not limited to: citrate buffers, phosphate buffers, trisbuffers, borate buffers, lactate buffers, glycyl glycin buffers,arginine buffers, carbonate buffers, acetate buffers, glutamate buffers,ammonium buffers, glycin buffers, alkylamine buffers, aminoethyl alcoholbuffers, ethylenediamine buffers, tri-ethanol amine, imidazole buffers,pyridine buffers and barbiturate buffers and mixtures thereof (cf.Remington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co.,Easton, Pa., 1990, or Remington: The Science and Practice of Pharmacy,19th Edition (1995), or handbooks from Amersham-Pharmacia Biotech).

The choice of starting pH, buffer and ionic strength is done accordingto well-known techniques such as conventional test-tube methods, cf.e.g. handbooks from Amersham-Pharmacia Biotech. The chromatographic ionexchange resin is chosen depending on the specific peptide to bepurified and the conditions employed, such as pH, buffer, ionic strengthetc., which are known to the person skilled in the art (that is,typically, pH below the isoelectric point (pI) of the peptide for cationexchange resins and pH above pI of the peptide for anion exchangeresins, a sufficient buffer strength to maintain the desired pH, and asufficiently low ionic strength possibly induced by the saltconcentration), and includes but is not limited to Sepharose resins,Sephadex resins, Streamline resins, and Source resins fromAmersham-Pharmacia Biotech, HyperD resins, Trisacryl resins, andSpherosil resins from BioSepra, TSKgel resins and Toyopearl resins fromTosoHaas, Fractogel EMD resins from Merck, Poros resins from PerseptiveBiosystems, Macro-Prep resins from BioRAD, Expression resins fromWhatman etc.

The term “a solution consisting essentially of an organic modifier,water, optionally a salt component and optionally a buffer” as usedherein, is intended to mean a solution containing one or more organicmodifiers, water, one or more salt components or no salt component andone or more buffers or no buffer, and optionally one or more furtherconventional components which the person skilled in the art wouldconsider adding, according to conventional ion exchange chromatographyprocesses.

The term “related impurities” as used herein, is intended to mean one ormore impurities with a different local or overall net charge from thepeptide, for instance truncated forms, all kinds of extended forms(extra amino acids, various derivatives including esters etc.),deamidated forms, incorrectly folded forms, forms with undesiredglycosylation including sialylation, “lack of γ-carboxy glutamic acid”,and others, as long as they elute before the peptide.

The term “pH-values”, as used herein in connection with “at pH-values”,“at the same or lower pH-values” and “at the same or higher pH-values”,is intended to mean that the pH-value in step a) may be constant or mayvary, typically, within 3 pH units, and subsequently, the pH-value instep b) may be at the same constant or varying pH-value as in step a),or may be at a different pH-value, which may be constant or may vary,typically, within 3 pH units. Such pH-values may typically be maintainedor varied by addition of a buffer and/or an inorganic or organic acid orbase, e.g. HCl, NaOH, H₂O, acetic acid, NH₃, KOH, H₂SO₄, citric acid.

The term “lower” as used herein, in connection with related impuritieshaving a local or overall negative (or positive) net charge which islower than the negative (or positive) net charge of the peptide, isintended to mean that the numeric value of the local or overall netcharge of the related impurities is lower than the numeric value of thelocal or overall net charge of said peptide.

The present invention also relates to the following aspects:

Aspect 1. A cation exchange chromatography process for purifying apeptide from a mixture comprising said peptide and related impurities,comprising the steps of:

a) eluting said related impurities of said mixture in a solutionconsisting essentially of an organic modifier, water, optionally a saltcomponent and optionally a buffer, at a linear or step gradient orisocratically in salt component, and at pH-values optionally maintainedwith a buffer so that said peptide has a positive local or overall netcharge and said related impurities have a local or overall positive netcharge which is lower than the positive net charge of said peptide so asto remove said related impurities,b) subsequently, eluting said peptide by a step or linear change to anaqueous solvent optionally with a salt component, at the same or higherpH-values optionally maintained with a buffer.

Aspect 2. An anion exchange chromatography process for purifying apeptide from a mixture comprising said peptide and related impurities,comprising the steps of:

a) eluting said related impurities of said mixture in a solutionconsisting essentially of an organic modifier, water, optionally a saltcomponent and optionally a buffer, at a linear or step gradient orisocratically in salt component, and at pH-values optionally maintainedwith a buffer so that said peptide has a negative local or overall netcharge and said related impurities have a local or overall negative netcharge which is lower than the negative net charge of said peptide so asto remove said related impurities,b) subsequently, eluting said peptide by a step or linear change to anaqueous solvent optionally with a salt component, at the same or lowerpH-values optionally maintained with a buffer.

Aspect 3. The process according to aspect 1 or 2 wherein the ratio oforganic modifier to water on a weight percent basis is from 1:99 to99:1.

Aspect 4. The process according to any one of aspects 1–3 wherein saidorganic modifier is selected from C₁₋₆-alkanol, C₁₋₆-alkenol orC₁₋₆-alkynol, urea, guanidine, or C₁₋₆-alkanoic acid, C₂₋₆-glycol, orC₃₋₇-polyalcohol including sugars.

Aspect 5. The process according to any one of aspects 14 wherein saidsalt component in step a) is selected from any organic or inorganicsalt, preferably NaCl, KCl, NH₄Cl, CaCl₂, sodium acetate, potassiumacetate, ammonium acetate, sodium citrate, potassium citrate, ammoniumcitrate, sodium sulphate, potassium sulphate, ammonium sulphate, calciumacetate or mixtures thereof.

Aspect 6. The process according to any one of aspects 14 wherein no saltcomponent is present in step a.

Aspect 7. The process according to any one of aspects 1–5 wherein saidsalt component gradient in step a) is a step or linear salt componentgradient.

Aspect 8. The process according to aspects 7 wherein said salt componentis present in a concentration selected from the range of 0.1 mmol/kg to3000 mmol/kg.

Aspect 9. The process according to any one of aspects 1–8 wherein saidsalt component in step b) is selected from any organic or inorganicsalt, preferably NaCl, KCl, NH₄Cl, CaCl₂, sodium acetate, potassiumacetate, ammonium acetate, sodium citrate, potassium citrate, ammoniumcitrate, sodium sulphate, potassium sulphate, ammonium sulphate, calciumacetate or mixtures thereof.

Aspect 10. The process according to any one of aspects 1–9 wherein instep b) said salt component is present in a concentration selected fromthe range of 0.1 mmol/kg to 3000 mmol/kg.

Aspect 11. The process according to any one of aspects 1–8 wherein nosalt component is present in step b.

Aspect 12. The process according to any one of aspects 1–11 wherein saidbuffer in step a) or b) is independently selected from citrate buffers,phosphate buffers, tris buffers, borate buffers, lactate buffers, glycylglycin buffers, arginine buffers, carbonate buffers, acetate buffers,glutamate buffers, ammonium buffers, glycin buffers, alkylamine buffers,aminoethyl alcohol buffers, ethylenediamine buffers, tri-ethanol amine,imidazole buffers, pyridine buffers and barbiturate buffers and mixturesthereof.

Aspect 13. The process according to any one of aspects 1–12 wherein saidbuffer in step a) is present in a concentration selected from the rangeof 0.1 mmol/kg to 500 mmol/kg.

Aspect 14. The process according to any one of aspects 1–13 wherein saidbuffer in step b) is present in a concentration selected from the rangeof 0.1 mmol/kg to 1000 mmol/kg.

Aspect 15. The process according to any one of aspects 1–11 wherein nobuffer is present in step a).

Aspect 16. The process according to any one of aspects 1–11 wherein nobuffer is present in step b).

Aspect 17. A method for isolating a peptide, the method includingpurification of a peptide from a mixture containing said peptide andrelated impurities via a cation exchange chromatography process, thecation exchange chromatography process comprising the steps of:

-   -   a) eluting said related impurities of said mixture in a solution        consisting essentially of an organic modifier, water, optionally        a salt component and optionally a buffer, at a linear or step        salt component gradient or isocratically, and at pH-values        optionally maintained with a buffer so that said peptide has a        positive local or overall net charge and said related impurities        have a local or overall positive net charge which is lower than        the positive net charge of said peptide so as to remove said        related impurities,    -   b) subsequently, eluting said peptide by a step or linear change        to an aqueous solvent optionally with a salt component, at the        same or higher pH-values optionally maintained with a buffer;        and subsequently, if necessary, subjecting to analytical tests        and/or further purification, and isolating said peptide in a        conventional manner.

Aspect 18. A method for isolating a peptide, the method includingpurification of a peptide from a mixture containing said peptide andrelated impurities via an anion exchange chromatography process, theanion exchange chromatography process comprising the steps of:

-   -   a) eluting said related impurities of said mixture in a solution        consisting essentially of an organic modifier, water, optionally        a salt component and optionally a buffer, at a linear or step        salt component gradient or isocratically, and at pH-values        optionally maintained with a buffer so that said peptide has a        negative local or overall net charge and said related impurities        have a local or overall negative net charge which is lower than        the negative net charge of said peptide so as to remove said        related impurities,    -   b) subsequently, eluting said peptide by a step or linear change        to an aqueous solvent optionally with a salt component, at the        same or lower pH-values optionally maintained with a buffer;        and subsequently, if necessary, subjecting to analytical tests        and/or further purification, and isolating said peptide in a        conventional manner.

Aspect 19. The process or method according to any one of aspects 1–18wherein said peptide to be purified is selected from polypeptides,oligopeptides, proteins, receptors, vira, as well as homologues,analogues and derivatives thereof, preferably glucagon, hGH, insulin,aprotinin, FVII, TPA, FVIIa, FFR-FVIIa, heparinase, ACTH, HeparinBinding Protein, corticotropin-releasing factor, angiotensin,calcitonin, insulin, glucagon-like peptide-1, glucagon-like peptide-2,insulin-like growth factor-1, insulin-like growth factor-2, fibroblastgrowth factors, gastric inhibitory peptide, growth hormone-releasingfactor, pituitary adenylate cyclase activating peptides, secretin,enterogastrin, somatostatin, somatotropin, somatomedin, parathyroidhormone, thrombopoietin, erythropoietin, hypothalamic releasing factors,prolactin, thyroid stimulating hormones, endorphins, enkephalins,vasopressin, oxytocin, opiods, DPP IV, interleukins, immunoglobulins,complement inhibitors, serpin protease inhibitors, cytokines, cytokinereceptors, PDGF, tumor necrosis factors, tumor necrosis factorsreceptors, growth factors, GIP, exendins, peptide histidine-methionineamide, helospectins, helodermin, pituitary adenylate cyclase activatingpeptide-related peptide, vasoactive intestinal polypeptide and analoguesas well as derivatives thereof, more preferably glucagon, hGH, insulin,aprotinin, FVII, FVIIa, FFR-FVIIa, heparinase, glucagon-like peptide-1,glucagon-like peptide-2 and analogues as well as derivatives thereof,such as Arg³⁴GLP-1(7-37), human insulin, and B28IsoAsp insulin.

EXAMPLES

The present invention is further illustrated by the following exampleswhich, however, are not to be construed as limiting the scope ofprotection. The features disclosed in the foregoing description and inthe following examples may, both separately and in any combinationthereof, be material for realising the invention in diverse formsthereof.

Example 1

Arg³⁴GLP-1(7-37) was expressed in yeast (Sacch. cerevisiae) byconventional recombinant DNA technology e.g as described in WO 98/08871.Arg³⁴GLP-1(7-37) fermentation broth was purified by a conventionalreverse phase chromatography capture step, and subsequently precipitatedat the pI (isoelectric point) of Arg³⁴GLP-1(7-37). 2 g of theprecipitate containing Arg³⁴GLP-1(7-37) and the truncated form missing ahistidine and an alanine residue, Arg³⁴GLP-1(9-37), as one of severalimpurities was suspended in 100 ml water and dissolved by pH adjustmentto 8.2. The resulting mixture was adjusted to pH 3.4 and filtered. 21 mlof the filtrate was applied to a 20 ml Source 30S (Amersham PharmaciaBiotech) column equilibrated with 100 ml 20 mmol/kg citric acid, 75mmol/kg KCl, 45% w/w ethanol, pH 3.5. The truncated form waseluted/washed off by a step gradient of 60 ml 20 mmol/kg citric acid,87.5 mmol/kg KCl, 45% w/w ethanol, pH 3.5. The target peptide,Arg³⁴GLP-1(7-37), was eluted in a single peak by a step gradient of 100ml 200 mmol/kg Tris-hydroxymethyl amino-methane, pH 8.5. A chromatogramis shown in FIG. 1.

RP-HPLC analysis for identification/verification of collected peaks wascarried out on a C₁₈-substituted 120 Å silica (YMC) 4.0×250 mm columnwith 5 μm particles. Buffer A consisted of 0.15 M (NH₄)₂SO₄ in 7.8%acetonitrile, pH 2.5, and buffer B contained 63.4% acetonitrile. Lineargradients from 37–41% B in 12 min followed by 41–100% B in 15 min wererun at a flow rate of 1 ml/min. The chromatographic temperature was keptat 60° C. and UV detection was performed at 214 nm. Analytical resultswere:

Arg³⁴GLP-1- Arg³⁴GLP-1- (7-37)content (9-37)content Sample forapplication 48% 18% Wash containing ethanol  1% 76% Aqueous eluate 65% 1%Chromatograms of the sample for application and the eluate is shown inFIGS. 2 and 3, respectively. The analytical results show a selectiveremoval of the truncated form by the wash step containing ethanol and ahigh reduction of the truncated form in the aqueous eluate by employmentof the cation exchange chromatography method.

Example 2

Arg³⁴GLP-1(7-37) fermentation broth was purified by cation exchangechromatography and precipitated as described in Example 1.

2 g of the precipitate containing Arg³⁴GLP-1 (7-37) and the truncatedform, Arg³⁴GLP-1 (9-37), as one impurity was suspended and dissolved in100 ml 20 mmol/kg glycin, pH 9.0 resulting in a final pH of 8.4. Theresulting mixture was adjusted to pH 3.5 and filtered. 52 ml of thefiltrate was applied to a 20 ml Source 30S (Amersham Pharmacia Biotech)column equilibrated with 60 ml 20 mmol/kg citric acid, 75 mmol/kg KCl,45% w/w ethanol, pH 3.5. The truncated form was eluted/washed off by astep gradient of 60 ml 20 mmol/kg citric acid, 87.5 mmol/kg KCl, 45% w/wethanol, pH 3.5. The target peptide, Arg³⁴GLP-1(7-37), was eluted in asingle peak by a step gradient of 100 ml 200 mmol/kg glycin, pH 9.0.RP-HPLC analysis for identification/verification of collected peaks wascarried out as described in Example 1. The analytical results show aselective removal of the truncated form by the wash step containingethanol and a high reduction of the truncated form in the aqueous eluateby employment of the cation exchange chromatography method.

Example 3

Arg³⁴GLP-1(7-37) fermentation broth was purified by a conventionalcation exchange chromatography capture step followed by a conventionalRP-HPLC purification step. 4 volumes of water was added to the RP-HPLCpool containing Arg³⁴GLP-1 (7-37) and the truncated form,Arg³⁴GLP-1(9-37), as an impurity. 175 ml of the resulting solution wasadjusted to pH 3.5 and applied to a 20 ml Source 30S (Amersham PharmaciaBiotech) column equilibrated with 100 ml 20 mmol/kg citric acid, 75mmol/kg KCl, 45% w/w ethanol, pH 3.5. The column was washed with 20 mlequilibration solution, and the truncated form was eluted/washed off bya linear gradient from 75 to 100 mmol/kg KCl (20 mmol/kg citric acid,45% w/w ethanol, pH 3.5). The target peptide, Arg³⁴GLP-1(7-37), waseluted in a single peak by a step gradient of 100 ml 100 mmol/kg Na₂CO₃,pH 9.5. A chromatogram is shown in FIG. 4.

Example 4

Arg³⁴GLP-1 (7-37) was expressed, captured by cation exchange, andpurified by RP-HPLC as described in Example 3.

1 volume of water was added to the RP-HPLC pool containingArg³⁴GLP-1(7-37) and the truncated form, Arg³⁴GLP-1(9-37), as animpurity. 86 ml of the resulting solution was adjusted to pH 3.5 andapplied to a 20 ml Source 30S (Amersham Pharmacia Biotech) columnequilibrated with 100 ml 20 mmol/kg citric acid, 75 mmol/kg KCl, 45% w/wethanol, pH 3.5. The column was washed with 20 ml equilibrationsolution, and the truncated form was eluted/washed off by a lineargradient from 75 to 100 mmol/kg KCl (20 mmol/kg citric acid, 45% w/wethanol, pH 3.5). The target peptide, Arg³⁴GLP-1(7-37), was eluted by astep gradient of 100 ml 100 mmol/kg H₃BO₃, 100 mmol/kg NaCl, pH 9.5.

Example 5

Arg³⁴GLP-1 (7-37) was isolated from the fermentation broth byconventional reverse phase chromatography and precipitated as describedin Example 1.

10 g of the precipitate containing Arg³⁴GLP-1(7-37) and the truncatedform, Arg³⁴GLP-1(9-37), as one of several impurities was suspended in500 ml water and dissolved by pH adjustment to 8.3 to a Arg³⁴GLP-1(7-37)concentration of approximately 1.6 mg/ml. 5 ml of the resulting solutionwas adjusted to pH 3.5 and applied to a 20 ml Source 30S (AmershamPharmacia Biotech) column equilibrated with 60 ml 0.42% w/w citric acid,34% w/w ethanol, pH 3.5. The truncated form was eluted/washed off by alinear gradient from 0 to 1.29% w/w KCl (0.42% w/w citric acid, 34% w/wethanol, pH 3.5). The target peptide, Arg³⁴GLP-1(7-37), was eluted in asingle peak by a step gradient of 100 ml 200 mmol/kg glycin, pH 9.5. Achromatogram is shown in FIG. 5.

Example 6

Arg³⁴GLP-1 (7-37) was isolated from the fermentation broth byconventional reverse phase chromatography and precipitated as describedin Example 1.

10 g of the precipitate containing Arg³⁴GLP-1(7-37) and the truncatedform, Arg³⁴GLP-1(9-37), as one of several impurities was suspended in500 ml water and dissolved by pH adjustment to 8.3 to a Arg³⁴GLP-1(7-37) concentration of approximately 1.6 mg/ml. 5 ml of the resultingsolution was adjusted to pH 3.2 and applied to a 20 ml Source 30S(Amersham Pharmacia Biotech) column equilibrated with 60 ml 0.42% w/wcitric acid, 29% w/w ethanol, pH 3.5. The truncated form waseluted/washed off by a linear gradient from 0 to 1.96% w/w KCl (0.42%w/w citric acid, 29% w/w ethanol, pH 3.5). The target peptide,Arg³⁴GLP-1(7-37), was eluted by a step gradient of 100 ml 200 mmol/kgglycin, pH 9.5.

Example 7

Arg³⁴GLP-1 (7-37) was isolated from the fermentation broth byconventional reverse phase chromatography and precipitated as describedin Example 1.

10 g of the precipitate containing Arg³⁴GLP-1(7-37) and the truncatedform, Arg³⁴GLP-1 (9-37), as one of several impurities was suspended in500 ml water and dissolved by pH adjustment to 8.3 to a Arg³⁴GLP-1(7-37) concentration of approximately 1.6 mg/ml. 5 ml of the resultingsolution was adjusted to pH 3.5 and applied to a 20 ml Source 30S(Amersham Pharmacia Biotech) column equilibrated with 60 ml 0.42% w/wcitric acid, 51% w/w ethanol, pH 3.5. The truncated form waseluted/washed off by a linear gradient from 0 to 1.00% w/w KCl (0.42%w/w citric acid, 51% w/w ethanol, pH 3.5). The target peptide,Arg³⁴GLP-1(7-37), was eluted by a step gradient of 100 ml 200 mmol/kgglycin, pH 9.5.

Example 8

Arg³⁴GLP-1 (7-37) was isolated from the fermentation broth byconventional reverse phase chromatography and precipitated as describedin Example 1.

10 g of the precipitate containing Arg³⁴GLP-1 (7-37) and the truncatedform, Arg³⁴GLP-1(9-37), as one of several impurities was suspended in500 ml water and dissolved by pH adjustment to 8.3 to a Arg³⁴GLP-1(7-37)concentration of approximately 1.6 mg/ml. 5 ml of the resulting solutionwas adjusted to pH 3.5 and applied to a 20 ml Source 30S (AmershamPharmacia Biotech) column equilibrated with 60 ml 0.42% w/w citric acid,71% w/w ethanol, pH 3.5. The truncated form was eluted/washed off by alinear gradient from 0 to 0.48% w/w KCl (0.42% w/w citric acid, 71% w/wethanol, pH 3.5). The target peptide, Arg³⁴GLP-1(7-37), was eluted by astep gradient of 100 ml 200 mmol/kg glycin, pH 9.5.

Example 9

Arg³⁴GLP-1(7-37) was isolated from the fermentation broth byconventional reverse phase chromatography and precipitated as describedin Example 1.

10 g of the precipitate containing Arg³⁴GLP-1(7-37) and the truncatedform, Arg³⁴GLP-1(9-37), as one of several impurities was suspended in500 ml water and dissolved by pH adjustment to 8.3 to a Arg³⁴GLP-1(7-37) concentration of approximately 1.6 mg/ml. 5 ml of the resultingsolution was adjusted to pH 3.5 and applied to a 20 ml Source 30S(Amersham Pharmacia Biotech) column equilibrated with 60 ml 0.42% w/wcitric acid, 40% w/w 2-propanol, pH 3.5. The truncated form waseluted/washed off by a linear gradient from 0 to 0.61% w/w KCl (0.42%w/w citric acid, 40% w/w 2-propanol, pH 3.5). The target peptide,Arg³⁴GLP-1(7-37), was eluted in a single peak by a step gradient of 100ml 200 mmol/kg glycin, pH 9.5. A chromatogram is shown in FIG. 6.

Example 10

Arg³⁴GLP-1 (7-37) was isolated from the fermentation broth byconventional reverse phase chromatography and precipitated as describedin Example 1.

10 g of the precipitate containing Arg³⁴GLP-1(7-37) and the truncatedform, Arg³⁴GLP-1(9-37), as one of several impurities was suspended in500 ml water and dissolved by pH adjustment to 8.3 to a Arg³⁴GLP-1(7-37) concentration of approximately 1.6 mg/ml. 5 ml of the resultingsolution was adjusted to pH 3.5 and applied to a 8 ml Poros 50 HS (PEBiosystems) column equilibrated with 24 ml 0.42% w/w citric acid, 51%w/w ethanol, pH 3.5. The truncated form was eluted/washed off by alinear gradient from 0 to 1.34% w/w KCl (0.42% w/w citric acid, 51% w/wethanol, pH 3.5). The target peptide, Arg³⁴GLP-1(7-37), was eluted by astep gradient of 40 ml 200 mmol/kg glycin, pH 9.5. A chromatogram isshown in FIG. 7.

Example 11

Arg³⁴GLP-1 (7-37) was isolated from the fermentation broth byconventional reverse phase chromatography and precipitated as describedin Example 1.

10 g of the precipitate containing Arg³⁴GLP-1(7-37) and the truncatedform, Arg³⁴GLP-1(9-37), as one of several impurities was suspended in500 ml water and dissolved by pH adjustment to 8.3 to a Arg³⁴GLP-1(7-37)concentration of approximately 1.6 mg/ml. 5 ml of the resulting solutionwas adjusted to pH 3.5 and applied to a 8 ml Poros 50 HS (PE Biosystems)column equilibrated with 24 ml 0.42% w/w citric acid, 40% w/w2-propanol, pH 3.5. The truncated form was eluted/washed off by a lineargradient from 0 to 1.34% w/w KCl (0.42% w/w citric acid, 40% w/w2-propanol, pH 3.5). The target peptide, Arg³⁴GLP-1 (7-37), was elutedby a step gradient of 40 ml 200 mmol/kg glycin, pH 9.5. A chromatogramis shown in FIG. 8.

Example 12

Arg³⁴GLP-1 (7-37) was isolated from the fermentation broth byconventional reverse phase chromatography and precipitated as describedin Example 1.

10 g of the precipitate containing Arg³⁴GLP-1(7-37) and the truncatedform, Arg³⁴GLP-1(9-37), as one of several impurities was suspended in500 ml water and dissolved by pH adjustment to 8.3 to a Arg³⁴GLP-1(7-37) concentration of approximately 1.6 mg/ml. 5 ml of the resultingsolution was adjusted to pH 3.5 and applied to a 20 ml Source 30S(Amersham Pharmacia Biotech) column equilibrated with 60 ml 0.42% w/wcitric acid, 40% w/w 2-methyl-2,4-pentanediol, pH 3.5. The truncatedform was eluted/washed off by a linear gradient from 0 to 0.60% w/w KCl(0.42% w/w citric acid, 40% w/w 2-methyl-2,4-pentanediol, pH 3.5). Thetarget peptide, Arg³⁴GLP-1(7-37), was eluted in a single peak by a stepgradient of 100 ml 200 mmol/kg glycin, pH 9.5. A chromatogram is shownin FIG. 9.

Example 13

A mixture of 7.7 mg/ml human insulin and 0.8 mg/ml human insulin ethylester (B30) was obtained by conventional methods as described elsewhere(cf. I. Mollerup, S. W. Jensen, P. Larsen, O. Schou, L. Snel: Insulin,Purification, in M. C. Flickinger, S. W. Drew: Encyclopedia ofBioprocess Technology: Fermentation, Biocatalysis, and Bioseparation,John Wiley & Sons, 1999). The mixture contained 4 mmol/l EDTA, 16% w/wethanol, pH 7.5. 2 ml of the mixture was applied to a 20 ml TSK-GelQ-5PW (TosoHaas) column equilibrated with 40 ml 0.15% w/wtriethanolamine, 42.5% w/w ethanol, pH 7.5. The human insulin ethylester impurity was eluted/washed off by a linear gradient from 0 to1.14% w/w sodium citrate tri-hydrate (0.15% w/w triethanolamine, 42.5%w/w ethanol, pH 7.5). The target protein, human insulin, was eluted in asingle peak by a step gradient of 60 ml 2.72% w/w sodium citratetri-hydrate, 0.15% w/w triethanolamine, pH 7.5. A chromatogram is shownin FIG. 10.

Example 14

A mixture of human insulin and human insulin ethyl ester (B30) wasobtained as described in Example 13. 2 ml of the mixture was applied toa 20 ml TSK-Gel Q-5PW (TosoHaas) column equilibrated with 40 ml 0.15%w/w triethanolamine, 42.5% w/w ethanol, pH 7.5. The human insulin ethylester impurity was eluted/washed off by a linear gradient from 0 to0.90% w/w sodium citrate tri-hydrate (0.15% w/w triethanolamine, 42.5%w/w ethanol, pH 7.5). The target protein, human insulin, was eluted in asingle peak by a step gradient of 60 ml 100 mmol/l citric acid, pH 3. Achromatogram is shown in FIG. 11.

Example 15

A mixture of human insulin and human insulin ethyl ester (B30) wasobtained as described in Example 13. 2 ml of the mixture was applied toa 20 ml TSK-Gel Q-5PW (Toso-Haas) column equilibrated with 40 ml 0.15%w/w triethanolamine, 71% w/w ethanol, pH 7.5. The human insulin ethylester impurity was eluted/washed off by a linear gradient from 0 to1.63% w/w sodium citrate tri-hydrate (0.15% w/w triethanolamine, 71% w/wethanol, pH 7.5). The target protein, human insulin, was eluted in asingle peak by a step gradient of 60 ml 2.72% w/w sodium citratetri-hydrate, 0.15% w/w triethanolamine, pH 7.5.

Example 16

A mixture of 9.0 mg/ml human insulin and 0.6 mg/ml human insulin ethylester (B30) was obtained as described in Example 13. The mixturecontained 4 mmol/l EDTA, pH 7.5 with a human insulin concentration of 9mg/ml. 2 ml of the mixture was applied to a 20 ml TSK-Gel Q-5PW(TosoHaas) column equilibrated with 40 ml 0.15% w/w triethanolamine, 81%w/w ethanol, pH 7.5. The human insulin ethyl ester impurity waseluted/washed off by a linear gradient from 0 to 2.18% w/w sodiumcitrate tri-hydrate (0.15% w/w triethanolamine, 81% w/w ethanol, pH7.5). The target protein, human insulin, was eluted in a single peak bya step gradient of 60 ml 2.72% w/w sodium citrate tri-hydrate, 0.15% w/wtriethanolamine, pH 7.5. A chromatogram is shown in FIG. 12.

Example 17

A mixture of human insulin and human insulin ethyl ester (B30) wasobtained as described in Example 16. 2 ml of the mixture was applied toa 20 ml TSK-Gel Q-5PW (TosoHaas) column equilibrated with 40 ml 0.15%w/w triethanolamine, 51% w/w ethanol, pH 7.5. The human insulin ethylester impurity was eluted/washed off by a linear gradient from 0 to1.09% w/w sodium citrate tri-hydrate (0.15% w/w triethanolamine, 51% w/wethanol, pH 7.5). The target protein, human insulin, was eluted in asingle peak by a step gradient of 60 ml 2.72% w/w sodium citratetri-hydrate, 0.15% w/w triethanolamine, pH 7.5.

1. A method for purifying a peptide from a mixture comprising saidpeptide and related impurities, said method comprising: a) eluting saidrelated impurities of said mixture from an anion exchange chromatographymatrix using a solution comprising an organic modifier, water, a buffer,and optionally a salt component at a linear or step gradient orisocratically in salt component, and at pH-values maintained with abuffer so that said peptide has a negative local or overall net chargeand said related impurities have a local or overall negative net chargewhich is lower than the negative net charge of said peptide so as toremove said related impurities; and without an intervening step, b)subsequently, eluting said peptide in the absence of an organicmodifier, by a step or linear change to an aqueous solvent optionallywith a salt component, at the same or lower pH-values maintained with abuffer.
 2. The method according to claim 1 further comprising subjectingthe peptide eluted in step (b) to analytical tests and/or furtherpurification.
 3. The method of claim 1, wherein said peptide to bepurified is selected from polypeptides, oligopeptides, proteins, andreceptors.
 4. The method of claim 1, wherein said peptide to be purifiedis selected from glucagon, hGH, insulin, FactorVII, FactorVIIa,FactorVIIai, FFR-FactorVIIa, glucagon-like peptide-1, glucagon-likepeptide-2 and analogs thereof.
 5. The method of claim 1, wherein theratio of organic modifier to water on a weight percent basis is from1:99 to 99:1.
 6. The method of claim 1, wherein the organic modifier isselected from C₁₋₆alkanol, C₁₋₆alkenol, C₁₋₆-alkynol, urea, guanidine,C₁₋₆-alkanoic acid, C₂₋₆-glycol, or C₃₋₇-polyalcohol.
 7. The methodaccording to claim 1, wherein the peptide is selected from the groupconsisting of Val⁸GLP-1(7-37), Thr⁸GLP-1(7-37), Met⁸GLP-1(7-37),Gly⁸GLP-1(7-37), Val⁸GLP-1(7-36) amide, Thr⁸GLP-1(7-36) amide,Met⁸GLP-1(7-36) amide, Gly⁸GLP-1(7-36) amide, Arg³⁴GLP-1(7-37, andB28IsoAsp insulin.
 8. An industrial method for producing a pure peptidefrom a mixture comprising said peptide and related impurities, saidmethod comprising: a) eluting said related impurities of said mixturefrom an anion exchange chromatography matrix using a solution comprisingan organic modifier, water, a buffer, and optionally a salt component ata linear or step gradient or isocratically in salt component, and atpH-values maintained with a buffer so that said peptide has a negativelocal or overall net charge and said related impurities have a local oroverall negative net charge which is lower than the negative net chargeof said peptide so as to remove said related impurities; and without anintervening step, b) subsequently, eluting said peptide in the absenceof an organic modifier, by a step or linear change to an aqueous solventoptionally with a salt component, at the same or lower pH-valuesmaintained with a buffer.
 9. The method according to claim 8 furthercomprising subjecting the peptide eluted in step (b) to analytical testsand/or further purification.
 10. The method according to claim 8,wherein said peptide to be purified is selected from polypeptides,oligopeptides, proteins, and receptors.
 11. The method according toclaim 8, wherein said peptide to be purified is selected from glucagon,hGH, insulin, FactorVII, FactorVIIa, FactorVIIai, FFR-FactorVIIa,glucagon-like peptide-1, glucagon-like peptide-2 and analogs thereof.12. The method according to claim 8, wherein the ratio of organicmodifier to water on a weight percent basis is from 1:99 to 99:1. 13.The method according to claim 8, wherein the organic modifier isselected from C₁₋₆alkanol, C₁₋₆alkenol, C₁₋₆alkynol, urea, guanidine,C₁₋₆-alkanoic acid, C₂₋₆-glycol, or C₃₋₇-polyalcohol.
 14. A method forpurifying a peptide selected from glucagon, hGH, insulin, FactorVII,FactorVIIa, FactorVIIai, FFR-FactorVIIa, glucagon-like peptide-1, andglucagon-like peptide-2 and analogs thereof from a mixture comprisingsaid peptide and related impurities, said method comprising: a) elutingsaid related impurities of said mixture from an anion exchangechromatography matrix using a solution comprising an organic modifier,water, a buffer, and optionally a salt component at a linear or stepgradient or isocratically in salt component, and at pH-values maintainedwith a buffer so that said peptide has a negative local or overall netcharge and said related impurities have a local or overall negative netcharge which is lower than the negative net charge of said peptide so asto remove said related impurities; and b) subsequently, eluting saidpeptide in the absence of an organic modifier, by a step or linearchange to an aqueous solvent optionally with a salt component, at thesame or lower pH-values maintained with a buffer.
 15. The methodaccording to claim 14, wherein said peptide is glucagon-like peptide-1or an analog thereof.
 16. The method according to claim 15, wherein saidglucagon-like peptide-1 analog is selected from the group consisting ofVal⁸GLP-1(7-37), Thr⁸GLP-1(7-37), Met⁸GLP-1(7-37), Gly⁸GLP-1(7-37),Val⁸GLP-1(7-36) amide, Thr⁸GLP-1(7-36) amide, Met⁸GLP-1(7-36) amide,Gly⁸GLP-1(7-36) amide and Arg³⁴GLP-1(7-37).
 17. The method according toclaim 14, wherein said peptide is insulin or an analog thereof.
 18. Themethod according to claim 17, wherein said insulin analog is B28IsoAspinsulin.
 19. The method according to claim 14, wherein the solution usedto elute related impurities from the anion exchange chromatographymatrix in step (a) includes a salt component.
 20. The method accordingto claim 19, wherein the related impurities are eluted from the anionexchange chromatography matrix in step (a) with a linear gradient in thesalt component.
 21. The method according to claim 19, wherein theorganic modifier in the solution used to elute related impurities fromthe anion exchange chromatography matrix in step (a) is a C₁–C₆ alkanol.